Bender J, Klein A
Molecular Genetics, Department of Biology, University of Marburg, D-35032, Marburg, Germany.
Nucleic Acids Res. 1997 Jul 15;25(14):2877-82. doi: 10.1093/nar/25.14.2877.
Model substrates mimicking the telomeric as well as the 5'-untranslated regions in front of a 5'-terminal part of a macronuclear gene of Euplotes crassus were transcribed in vitro using cellular extracts. The obtained transcripts were characterized by primer extension and shown to start at the natural initiation points. The situation changed in the absence of telomere binding protein or with substrates lacking functional telomeric sequences. In these cases non-specific transcription was observed. Since it had been previously found that transcription starts are frequently located at an apparently fixed distance from the telomere, a model substrate was constructed which carried a duplication of the non-transcribed region. This resulted in only one transcription start site, the position of which was conserved relative to the start of the open reading frame but moved away from the telomere by the distance of the duplication.
利用细胞提取物在体外转录模仿端粒以及粗尾真核生物大核基因5'末端部分前的5'非翻译区的模型底物。通过引物延伸对获得的转录本进行表征,并显示其在天然起始点开始。在没有端粒结合蛋白或底物缺乏功能性端粒序列的情况下,情况发生了变化。在这些情况下,观察到非特异性转录。由于先前已经发现转录起始点通常位于距端粒明显固定的距离处,因此构建了一个携带非转录区重复的模型底物。这导致只有一个转录起始位点,其位置相对于开放阅读框的起始是保守的,但通过重复的距离远离了端粒。